皮下预免疫卡介苗提高卡介苗膀胱灌注诱导的外周特异性免疫应答的实验研究

Experimental study of subcutaneous pre-immunization with BCG to improve the peripheral specific immune response upon intravesical BCG instillation

膀胱内灌注卡介苗(bacillus Calmette-Guerin,BCG)是经尿道膀胱肿瘤切除术(transurethral resection of bladder tumor,TURBt)后的中高危非肌层浸润性膀胱癌(non-muscle-invasive bladder cancer,NMIBC)患者的标准辅助治疗。然而,其作用机制仍不完全清楚。本研究利用小鼠皮下免疫模型和膀胱内灌注模型,使用多色流式染色技术检测膀胱内的免疫细胞浸润情况及外周纯蛋白衍生物(purified proteinderivate,PPD)特异性细胞免疫水平,探究皮下预免疫BCG对膀胱内BCG灌注诱导的外周特异性免疫应答的影响。研究结果显示,膀胱内BCG灌注3次后,小鼠脾脏中PPD特异性IFN-γ^(+)CD4^(+)T细胞(P<0.001)和CD8^(+)T细胞(P<0.05)的比例显著上升,并维持在一定的水平;同时,与单纯膀胱内BCG灌注组相比,BCG预免疫后再接受膀胱内BCG灌注的小鼠膀胱局部浸润的CD4^(+)T、NK和巨噬细胞显著增多。并且外周PPD特异性的IFN-γ^(+)、IL-2^(+)、TNF-α^(+)、IL-17A^(+)CD4^(+)T和CD8^(+)T细胞比例也显著上升,外周Ki67^(+)NK细胞的比例增高,杀伤能力显著增强。研究结果提示,膀胱内BCG灌注引起的局部免疫可以显著诱导外周PPD特异性T细胞免疫反应,经皮预免疫BCG后接受膀胱内BCG灌注的外周PPD特异性T细胞免疫反应的增强更为显著。

Intravesical bacillus Calmette-Guerin(BCG)instillation is a standard adjuvant treatment for patients with intermediate-risk and high-risk non-muscular-invasive bladder cancer(NMIBC)after transurethral resection of bladder tumor(TURBt).However,the mechanism of action has not been fully understood yet.In this study,a subcutaneous pre-immunization with BCG followed by intravesical instillation mouse model was established to explore the influence of subcutaneous BCG vaccination on the peripheral specific immune response upon intravesical BCG instillation.The infiltrating immune cells in the bladder and the peripheral purified protein derivate(PPD)specific cellular immune response were detected by multicolor flow cytometry.Our results firstly showed that the proportion of PPD-specific IFN-γ^(+)CD4^(+)T cells(P<0.001)and CD8^(+)T cells(P<0.05)in the spleens of mice receiving 3 times of intravesical BCG instillation were significantly increased and maintained at certain levels.When BCG were pre-immunized in the mice,the infiltration of CD4^(+)T cells,NK cells and macrophages in the bladder were dramatically elevated followed by intravesical BCG instillation when compared to those without BCG vaccination.In addition,the frequencies of PPD-specific IFN-γ^(+),IL-2^(+),TNF-α^(+),IL-17 A^(+)CD4^(+)T and CD8^(+)T cells,as well as the peripheral Ki67^(+)NK cells were remarkably increased.The killing ability was also prominently enhanced in the mice with percutaneous BCG vaccination.These results indicated that local immunity induced by intravesical BCG instillation could evoke peripheral PPD-specific cellular immune responses.Subcutaneous pre-immunization with BCG followed by intravesical BCG instillation could significantly improve the peripheral PPD-specific T cell immune responses followed by intravesical BCG instillation.